Abstract
Abstract: :
Purpose: Recent evidence suggests that nitric oxide (NO), one unconventional neurotransmitter, may be involved in lacrimal gland secretion. In this study, our goal was to determine the presence of NO in tears and lacrimal glands, determine whether NO is produced by lacrimal acinar cells, and explore the effect of exogenous NO on protein secretion. Methods: Tears were collected from the cul de sac by micropipette. Lacrimal glands were homogenized in PBS with a glass tissue grinder. Purified lacrimal acinar cells were cultured in Matrigel rafts for 4 days, then collected and incubated for 20 m without agonist or with carbachol (10-4 M) or sodium nitroprusside (SNP, 10-4 M). The supernatant culture media, tears, and homogenates were filtered using a 10 kDa cut-off filter. The filtrates were assayed for NO by measuring its end products, nitrate and nitrite, with a fluorometric assay kit and read with a GENios fluorescence microplate reader. Beta-hexosaminidase concentration was determined in the culture media by using 4-methylumbelliferyl N-acetyl-beta-D-glucosaminide as the substrate. Total protein concentrations were measured by the Coomassie blue method. Results: Total nitrate/nitrite concentrations were 374.35 µM in the tears and 225.34 µMole/kg tissue wet weight in lacrimal gland. Nitrate/nitrite were also detected in the supernatant culture media, and stimulation with 10-4 M carbachol had no significant effect on the concentration. SNP had no significant effect on beta-hexosaminidase or total protein secretion by the cultured lacrimal acinar cells, and carbachol-induced secretion appeared not to be influenced by SNP. Conclusions: Our data indicated that there were significant amounts of nitrate/nitrite in the tears and lacrimal glands. Cultured lacrimal acinar cells were capable of producing NO, and acute cholinergic stimulation appeared to have no influence on its rate of production. Exogenous NO had no significant effect on protein secretion by the cultured cells. These data are consistent with observations that neuronal NO synthase immunoreactivity was present in the mouse lacrimal gland. These data suggest that NO is not a direct secretagogue, but may influence lacrimal secretion indirectly by various mechanisms, such as modulating neurotransmitter release, influencing the rate of vascular perfusion through its action as a vasodilator, etc.
Keywords: lacrimal gland • nitric oxide • cornea: tears/tear film/dry eye